Changes of Ca2+ -ATPase Activities in Cell of Rice Seed lings During the Enhancement of Chilling Resistance Induced by Cold and Salt Pretreatment

The changes of Ca2+ -ATPase activities of plasmolemma, and tonoplast membrane in roots and leaf chloroplasts in rice ( Oryza sativa L. ) seedlings were investigated for exploring the mechanism of cross adaptation to different stresses in the plants during the enhancement of chilling resistance induced by cold and salt pretreatment. The results indicated that the chilling resistance of rice seedlings was enhanced markedly by cold and salt pretreatment, but this enhancement was inhibited by Ca2+-chelate ethyleneglycol-bis-(β-aminoethyl ether) N, N-tetraacetic acid (EGTA) and the calmodulin inhibitor chlorpromazine (CPZ), it showed the calcium messenger system was involved in the course of chilling resistance formation. The Ca2+ -ATPase activity of root plasmolemma and tonoplast membrane as well as the Fe(CN)63- reduction in root plasmolemma in nonpretreated seedlings were declined markedly during the chilling stress. The Ca2+ -ATPase activities of plasmolemma, tonoplast membrane and chloroplasts as well as the Fe(CN)63- reduction of plasmolemma were enhanced by cold pretreatment. The activities of Ca2+ -ATPase and Fe(CN)63- reduction of plasmolemma, as compared with nonpretreated seedlings has increased by 86.80% and 93.93% respectively. The effect of salt pretreatmerit on the Ca2+ -ATPase activities of plasmolemma and chloroplast as well as Fe(CN)63- reduction of plasmolemma were similar to the effect of cold pretreatment. Although the Ca2+ -ATPase activity of tonoplast membrane was declined by salt pretreatment, the activity was none the less markedly higher than that of the nonpretreated seedlings. It showed that there was stronger ability of maintaining calcium homeostasis in the seedlings following two pretreatment. The results displayed that the enhancement of chilling resistance in rice seedlings with cold and salt pretreatment might be related to the effective activation of Ca2+ -ATPase in two pretreatment seedlings, because the activated Ca2+ -ATPase could bring back rapidly the raised cytoplasmic Ca2+ concentration from chilling stress to the state of calcium homeostasis, leading to the maintenance of normal functioning of the calcium messenger system and physiological metabolism. It seems that the adapated mechanism to chilling stress in two seedlings with cold and salt pretreatment was similar.     

水稻幼苗根质膜氧化还原系统活性与抗冷性的关系

本试验以水稻幼苗为材料,研究冷胁迫和钙浸种、低温锻炼、低温锻炼结合钙浸种预处理分别对幼苗根质膜Ｆｅ（ＣＮ）３－６还原活性的影响。实验结果表明：冷胁迫降低了质膜Ｆｅ（ＣＮ）３－６还原活性;钙浸种、低温锻炼、低温锻炼结合钙浸种预处理均提高了质膜Ｆｅ（ＣＮ）３－６还源活性,尤其是削减了冷胁迫降低质膜Ｆｅ（ＣＮ）３－６还原活性的作用。根质膜Ｆｅ（ＣＮ）３－６还原活性与水稻幼苗抗冷力密切相关。     

Comparative proteomic analysis of early salt stress responsive proteins in roots and leaves of rice

Growth and productivity of rice (Oryza sativa L.) are severely affected by salinity. Understanding the mechanisms that protect rice and other important cereal crops from salt stress will help in the development of salt‐stress‐tolerant strains. In this study, rice seedlings of the same genetic species with various salt tolerances were studied. We first used 2DE to resolve the expressed proteome in rice roots and leaves and then used nanospray liquid chromatography/tandem mass spectrometry to identify the differentially expressed proteins in rice seedlings after salt treatment. The 2DE assays revealed that there were 104 differentially expressed protein spots in rice roots and 59 in leaves. Then, we identified 83 proteins in rice roots and 61 proteins in rice leaves by MS analysis. Functional classification analysis revealed that the differentially expressed proteins from roots could be classified into 18 functional categories while those from leaves could be classified into 11 functional categories. The proteins from rice seedlings that most significantly contributed to a protective effect against increased salinity were cysteine synthase, adenosine triphosphate synthase, quercetin 3‐O‐methyltransferase 1, and lipoxygenase 2. Further analysis demonstrated that the primary mechanisms underlying the ability of rice seedlings to tolerate salt stress were glycolysis, purine metabolism, and photosynthesis. Thus, we suggest that differentially expressed proteins may serve as marker group for the salt tolerance of rice.     

基于转录组测序的扎龙湿地野大麦耐盐性分析

通过转录组测序技术对扎龙湿地野大麦幼苗在200 mmol/L NaCl胁迫2 d后的叶片进行分析。对测序结果进行De novo拼接后,将差异表达基因在GO、KEGG数据库中进行比对注释。结果表明:NaCl胁迫2 d后,对照组与处理组分别获得Unigene序列61038个和46754个,检测到差异表达基因25465个,差异基因GO功能注释到3个大类的55个功能组。差异表达基因被注释到135个Pathway上,直观地显示出NaCl胁迫下野大麦幼苗体内发生调节及改变的代谢过程和信号通路,其中主要涉及光合作用、脯氨酸代谢、叶绿素代谢等途径。通过对野大麦幼苗叶片转录组分析,发掘相关耐盐基因,有助于培育野大麦及其近缘作物新品种,并对揭示植物耐盐性分子机制及相关代谢途径具有重要意义。     

氧化胁迫对水稻幼苗抗冷力的影响

利用Ｈ２Ｏ２和甲基紫精（ＭＶ）对水稻幼苗作三种不同程度的氧化胁迫预处理。结果表明：轻度氧化胁迫预处理（１０ｕｍｏｌ／ＬＨ２Ｏ２或１０ｕｍｏｌ／ＬＭＶ处理４ｈ）提高了水稻幼苗的抗冷力,严重氧化胁迫预处理（１０ｕｍｏｌ／ＬＨ２Ｏ２或１０ｕｍｏｌ／ＬＭＶ分别处理１６ｈ和４０ｈ）则削弱水稻幼苗的抗冷力。氧化胁迫预处理刺激了水稻幼苗叶片抗氧化酶（ＳＯＤ,ＣＡＴ,ＰＯＸ和ＡＰＸ）的活性。经冷胁迫后,不同预处理苗的叶片抗氧化酶活性、膜脂过氧化和膜结构的变化趋势不同：轻度氧化胁迫预处理使幼苗仍保持较高的抗氧化酶活性,减轻了由冷胁迫引起的膜脂过氧化和细胞膜的渗漏程度,而严重氧化胁迫预处理则相反。因此,水稻幼苗对氧化胁迫感知并作出反应的机制（氧化应激机制）在水稻幼苗对低温反应和适应过程中起着很重要的调节作用。     

冷锻炼和ABA诱导水稻幼苗提高抗冷性期间膜保护系统的变化

冷锻炼和ＡＢＡ处理提高了水稻幼苗叶绿体ＳＯＤ和ＧＲ活性及叶片抗氧化剂ＡｓＡ和ＧＳＨ的含量,降低了膜电解质泄漏,增强了幼苗的抗冷性．等电聚焦电泳分析表明,冷锻炼和ＡＢＡ处理苗叶绿体ＳＯＤ三条同工酶带和ＧＲ１、２、３和６同工酶带都有不同程度的增强．低温胁迫后,处理和未处理首的ＳＯＤ、ＧＲ活性和ＡＳＡ、ＧＳＨ含量均有所下降．但处理苗的水平仍维持在未处理苗之上．亚胺环已酮可抑制因冷锻炼和ＡＢＡ诱导增加的ＳＯＤ和ＧＲ活性,并使叶片电解质泄漏增大．本试验结果表明冷锻炼或ＡＢＡ诱导水稻幼苗抗冷性提高时,对防御活性氧的保护系统有类似的影响。     

不同胁迫预处理提高水稻幼苗抗寒性期间膜保护系统的变化比较

通过比较盐、冷和热激三种不同胁迫预处理提高水稻(Oryza sativa L.)抗寒性过程中膜保护系统的变化,探讨植物交叉适应机理。结果表明,水稻幼苗经不同胁迫预处理均可提高幼苗的抗寒性。与未预处理苗相比,不同胁迫(冷、热、盐)预处理之间在处理后、低温伤害后及恢复2d后的3个不同时期膜酶促和非酶促保护系统及同工酶酶谱变化各有异同,既有部分共性,也有其独特性。     

东乡野生稻苗期响应低温胁迫的转录组分析

为了解东乡野生稻（Oryza rufipogon）对低温胁迫的响应机制,对苗期的RNA-seq转录表达谱进行了研究。结果表明,与对照相比,共检测到10 200个差异表达基因（DEGs）,其中5 201个上调表达,4 999个下调表达,其中有426个DEGs位于已报道的水稻耐冷QTL区间,且37个为耐冷调控相关的家族基因。GO功能分类和KEGG代谢路径分析表明,核酸结合转录因子活性、氨基酸生物合成以及光合作用代谢等均参与响应低温胁迫过程。实时荧光定量分析表明,ABA响应蛋白基因、MYB转录因子和40S核糖体蛋白SA基因等12个可能与低温胁迫响应相关的DEGs表达模式与RNA-seq的一致。可见,植物激素传导途径和转录因子相关调控基因在东乡野生稻苗期响应低温胁迫过程中起重要作用。     

Comparative proteomic and metabolomic analyses reveal mechanisms of improved cold stress tolerance in bermudagrass (Cynodon dactylon (L.) Pers.) by exogenous calcium

As an important second messenger, calcium is involved in plant cold stress response, including chilling (<20 °C) and freezing (<0 °C). In this study, exogenous application of calcium chloride (CaCl₂) improved both chilling and freezing stress tolerances, while ethylene glycol‐bis‐(β‐aminoethyl) ether‐N,N,N,N‐tetraacetic acid (EGTA) reversed CaCl₂ effects in bermudagrass (Cynodon dactylon (L.) Pers.). Physiological analyses showed that CaCl₂ treatment alleviated the reactive oxygen species (ROS) burst and cell damage triggered by chilling stress, via activating antioxidant enzymes, non‐enzymatic glutathione antioxidant pool, while EGTA treatment had the opposite effects. Additionally, comparative proteomic analysis identified 51 differentially expressed proteins that were enriched in redox, tricarboxylicacid cycle, glycolysis, photosynthesis, oxidative pentose phosphate pathway, and amino acid metabolisms. Consistently, 42 metabolites including amino acids, organic acids, sugars, and sugar alcohols were regulated by CaCl₂ treatment under control and cold stress conditions, further confirming the common modulation of CaCl₂ treatment in carbon metabolites and amino acid metabolism. Taken together, this study reported first evidence of the essential and protective roles of endogenous and exogenous calcium in bermudagrass response to cold stress, partially via activation of the antioxidants and modulation of several differentially expressed proteins and metabolic homeostasis in the process of cold acclimation.     

Genetic engineering of rice capable of synthesizing fructans and enhancing chilling tolerance

Fructans are water-soluble fructose oligomers and polymers thatare based on sucrose, and have been implicated in protectingplants against water stress. Rice (Oryza sativa L.) is highlysensitive to chilling temperatures, and is not able to synthesizefructans. Two wheat fructan-synthesizing enzymes, sucrose:sucrose1-fructosyltransferase, encoded by wft2, or sucrose:fructan6-fructosyltransferase, encoded by wft1, were introduced intorice plants, and rice transformants that accumulate fructanswere successfully obtained. The mature leaf blades of transgenicrice lines with wft2 or wft1 accumulated 16.2 mg g^–1 FWof oligo- and polysaccharides mainly composed of inulin oligomersof more than DP7, and 3.7 mg g^–1 FW of oligo- and polysaccharides,mainly composed of phlein oligomers of more than DP15, respectively.The transgenic rice seedlings with wft2 accumulated significantlyhigher concentrations of oligo- and polysaccharides than non-transgenicrice seedlings, and exhibited enhanced chilling tolerance. Theoligo- and polysaccharide concentrations of seedlings expressingwft1 were obviously lower than those of lines expressing wft2,and no correlation between oligo- and polysaccharide concentrationsand chilling tolerance was detected in wft1-expressing ricelines. The results suggest that transgenic rice lines expressingwheat-derived fructosyltransferase genes accumulated large amountsof fructans in mature leaf blades and exhibited enhanced chillingtolerance at the seedling stage. This is the first report owingthat fructan accumulation enhanced tolerance to non-freezinglow temperatures. Key words: Chilling tolerance, fructan, fructosyltransferase, Oryza sativa, transgenic plant     

Locating QTLs controlling overwintering seedling rate in perennial glutinous rice 89-1 (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

A new cold tolerant germplasm resource named glutinous rice 89-1 (Gr89-1, Oryza sativa L.) can overwinter using axillary buds, with these buds being ratooned the following year. The overwintering seedling rate (OSR) is an important factor for evaluating cold tolerance. Many quantitative trait loci (QTLs) controlling cold tolerance at different growth stages in rice have been identified, with some of these QTLs being successfully cloned. However, no QTLs conferring to the OSR trait have been located in the perennial O. sativa L. To identify QTLs associated with OSR and to evaluate cold tolerance. 286 F₁₂ recombinant inbred lines (RILs) derived from a cross between the cold tolerant variety Gr89-1 and cold sensitive variety Shuhui527 (SH527) were used. A total of 198 polymorphic simple sequence repeat (SSR) markers that were distributed uniformly on 12 chromosomes were used to construct the linkage map. The gene ontology (GO) annotation of the major QTL was performed through the rice genome annotation project system. Three main-effect QTLs (qOSR2, qOSR3, and qOSR8) were detected and mapped on chromosomes 2, 3, and 8, respectively. These QTLs were located in the interval of RM14208 (35,160,202 base pairs (bp))–RM208 (35,520,147 bp), RM218 (8,375,236 bp)–RM232 (9,755,778 bp), and RM5891 (24,626,930 bp)–RM23608 (25,355,519 bp), and explained 19.6%, 9.3%, and 11.8% of the phenotypic variations, respectively. The qOSR2 QTL displayed the largest effect, with a logarithm of odds score (LOD) of 5.5. A total of 47 candidate genes on the qOSR2 locus were associated with 219 GO terms. Among these candidate genes, 11 were related to cell membrane, 7 were associated with cold stress, and 3 were involved in response to stress and biotic stimulus. OsPIP1;3 was the only one candidate gene related to stress, biotic stimulus, cold stress, and encoding a cell membrane protein. After QTL mapping, a total of three main-effect QTLs—qOSR2, qOSR3, and qOSR8—were detected on chromosomes 2, 3, and 8, respectively. Among these, qOSR2 explained the highest phenotypic variance. All the QTLs elite traits come from the cold resistance parent Gr89-1. OsPIP1;3 might be a candidate gene of qOSR2.